Abstract
Bovine β-casein (A2 genetic variant) was mutagenized to (L70S/P71S) and expressed in transgenic mouse milk. This protein now carries the signal (N68S69S70S71) that mimics a consensus eukaryotic glycosylation signal (N-X-S/T) (3). Hypothetically this protein should be glycosylated at N68 by any eukaryotic organism producing it. This novel protein was purified from transgenic mouse milk by Mono-S cation-exchange fast protein liquid chromatography (FPLC). The novel β-casein was separated without cross contamination from mouse caseins by using acetate buffer (pH 5.0) in the presence of 6 M urea, octyl-glucopyranoside and 2-β-mercaptoethanol. The purified (L70S/P71S) β-casein showed an N-linked oligosaccharide attached to Asn68 and different lectin binding profiles compared with the same protein expressed in yeast. The mouse-expressed β-casein (L70S/P71S) was specific to Concanavalin A, wheat germ agglutinin, Erythrina cristagalli agglutinin, and Ulex europaeus, indicating its oligosaccharide structure is different in the mammary gland of mouse than the reported glycosylated β-casein expressed in Pichia pastoris (4). In addition, the five serine residues located at amino terminus of wild type bovine β-casein were shown to be normally phosphorylated as in native bovine β-casein.
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